Mammals require molecular oxygen for essential metabolic processes including oxidative phosphorylation in which oxygen serves as electron acceptor during ATP formation. Systemic, local, and intracellular homeostatic responses elicited by hypoxia (the state in which oxygen demand exceeds supply) include erythropoiesis by individuals who are anemic or at high altitude (Jelkmann, Physiol. Rev. 72:449-489, 1992), neovascularization in ischemic myocardium; White et al., Circ. Res. 71:1490-1500, 1992), and glycolysis in cells cultured at reduced oxygen tension (Wolfe et al., Eur. J. Biochem. 135:405-412, 1983). These adaptive responses either increase oxygen delivery or activate alternate metabolic pathways that do not require oxygen. Hypoxia-inducible gene products that participate in these responses include erythropoietin (EPO) (reviewed in Semenza, Hematol. Oncol. Clinics N. Amer. 8:863-884, 1994), vascular endothelial growth factor (VEGF) (Shweiki et al., Nature 359:843-845, 1992; Banai et al., Cardiovasc. Res. 28:1176-1179, 1994; Goldberg & Schneider, J. Biol. Chem. 269:4355-4359, 1994), and glycolytic enzymes (Firth et al., Proc. Natl. Acad. Sci. USA 91:6496-6500, 1994; Semenza et al., J. Biol. Chem. 269:23757-23763, 1994).
The induction of HIF-1α activity by 1% oxygen was detected in many mammalian cell lines (Wang & Semenza, Proc. Natl. Acad. Sci. USA 90:4304-4308, 1993). The EPO enhancer directed hypoxia-inducible transcription of reporter genes transfected into non-EPO-producing cells (Maxwell et al., Proc. Natl. Acad. Sci. USA 90:2423-2427, 1993). RNAs encoding several glycolytic enzymes were induced by 1% oxygen. These experiments support the role of HIF-1α in activating homeostatic responses to hypoxia.
Hypoxia inducible factor-1 (HIF-1) is a mammalian transcription factor expressed uniquely in response to physiologically relevant levels of hypoxia (Wang, G. L., et al., Proc. Natl. Acad. Sci. USA 92:5510-5514, 1995; Wang, G. L., and Semenza, G. L., J. Biol. Chem. 270:1230-1237, 1995; U.S. Pat. No. 5,882,914). HIF-1 is a basic helix loop-helix protein that binds to cis-acting hypoxia-responsive elements of genes induced by hypoxia (Wang, G. L., and Semenza, G. L., Curr. Opin. Hematol. 3:156-162, 1992; Jiang, B. H., et al., J. Biol. Chem. 212:19253-19260, 1997). The genes that are activated by HIF-1 in cells subjected to hypoxia include EPO, vascular endothelial growth hormone (VEGF), heme oxygenase-1, inducible nitric oxide synthase, and glycolytic enzymes aldolase A, enolase 1, lactate dehydrogenase A, phosphofructokinase I, and phosphoglycerate kinase 1 (Semenza, G. L., et al., Kid. Int. 51:553-555, 1997). HIF-1 DNA binding activity and HIF-1 protein concentration increase exponentially as cells are subjected to decreasing oxygen concentrations (Jiang, B. H., et al., Am J. Physiol. 271:C 172-C1180, 1996).
The Hypoxia-Inducible transcription Factor 1α (HIF-1α) has been critically implicated in fundamental and pathophysiological mechanisms of regulation of metabolism and functions of many different types of cells and tissues. The adaptation of mammalian cells to low oxygen conditions is mediated in large part by the transcriptional induction of gene expression. Hypoxia-inducible factor (HIF) is crucial in the transcriptional response of cells to hypoxia. (Semenza G. Nat Rev Cancer 2003, 3, p. 721; Giaccia A, Siim B G, Johnson R S. HIF-1 as a target for drug development. Nat Rev Drug Discov. October 2003; 2(10):803-11).
HIF-1 stability and activity are regulated by post-translational modifications, chaperone function and alternative splicing. Hypoxia-inducible factor (HIF-1) is an oxygen-dependent transcriptional activator, which plays crucial roles in the angiogenesis of tumors and mammalian development. HIF-1 consists of a constitutively expressed HIF-1β subunit and one of three subunits (HIF-1α, HIF-2α or HIF-3α). The stability and activity of HIF-1α are regulated by various post-translational modifications, hydroxylation, acetylation, and phosphorylation. Therefore, HIF-1α interacts with several protein factors including PHD, pVHL, ARD-1, and p300/CBP. Under normoxia, the HIF-1α subunit is rapidly degraded via the von Hippel-Lindau tumor suppressor gene product (pVHL)-mediated ubiquitin-proteasome pathway. The association of pVHL and HIF-1α under normoxic conditions is triggered by the hydroxylation of prolines and the acetylation of lysine within a polypeptide segment known as the oxygen-dependent degradation (ODD) domain. On the contrary, in the hypoxia condition, HIF-1α subunit becomes stable and interacts with coactivators such as p300/CBP to modulate its transcriptional activity. Eventually, HIF-1 acts as a master regulator of numerous hypoxia-inducible genes under hypoxic conditions.
The target genes of HIF-1α are especially related to angiogenesis, cell proliferation/survival, and glucose/iron metabolism. Moreover, it was reported that the activation of HIF-1α is closely associated with a variety of tumors and oncogenic pathways. New evidence suggests that at least two members of the family of hypoxia-inducible factor (HIF) prolyl hydroxylases that regulate HIF stability in response to oxygen availability are themselves also targeted for proteosome-dependent degradation by the E3 ubiquitin ligases Siah1a and Siah2. (Nakayama K, Frew I J, Hagensen M, Skals M, Habelhah H, Bhoumik A, Kadoya T, Erdjument-Bromage H, Tempst P, Frappell P B, Bowtell D D, Ronai Z., Siah2 regulates stability of prolyl-hydroxylases, controls HIF1α abundance, and modulates physiological responses to hypoxia. Cell. Jun. 25, 2004; 117(7):851-3.)
HIF-1α activities in myeloid cells are considered to play a pro-inflammatory role since the HIF-1α is required for the inflammatory effects of cells of the innate immune system (Cramer T, Yamanishi Y, Clausen B E, Forster I, Pawlinski R, Mackman N, Haase V H, Jaenisch R, Corr M, Nizet V, Firestein G S, Gerber H P, Ferrara N and Johnson R S. HIF-1alpha is essential for myeloid cell-mediated inflammation. Cell 112: 645-657, 2003.).
The inflamed local tissue environments are hypoxic and the tissue damage-associated hypoxia is conducive to accumulation of elevated levels of extracellular adenosine. The recently provided genetic evidence for the critical role of extracellular adenosine and of Gs protein coupled A2A adenosine receptors in down-regulation of activated immune cells in vivo (Ohta A and Sitkovsky M. Role of G-protein-coupled adenosine receptors in downregulation of inflammation and protection from tissue damage. Nature 414: 916-920, 2001) suggested that inflammation-induced, local tissue damage-associated hypoxia and oxygen sensors may serve as primary signals of excessive tissue damage in order to de-activate immune cells.